Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp2 mutants preferentially undergo myelomonocytic differentiation and expansion despite being subjected to conditions that normally support only mast cell development. Evaluation of hematopoietic cell-specific transcription factor expression using quantitative RT-PCR indicates equal expression of PU.1 in mutant Shp2- and WT Shp2-expressing hematopoietic progenitors; however, GATA2 expression is substantially reduced and c-Jun expression is increased in mutant Shp2-expressing progenitors. Based on these findings, we hypothesized that mutant Shp2-induced Ras hyperactivation produces constitutive c-Jun expression permitting, as a co-activator of PU.1, excessive monocytic differentiation and reduced GATA2 expression. As a corollary, we hypothesized that ectopic expression of GATA2, but not of GATA2 lacking the C-terminal zinc finger (amino acids 330 – 407, DGATA2), which is needed to compete with c-Jun for binding to PU.1, would normalize mutant Shp2-induced monocytic differentiation and expansion. To examine this hypothesis, we utilized retroviral co-transduction of bone marrow Lin-enriched cells to generate six experimental groups:

  1. pMIEG3-Shp-2WT plus pCD4 (empty vector);

  2. pMIEG3-Shp2WT plus pCD4-GATA2;

  3. pMIEG3-Shp2WT plus pCD4-ΔGATA2;

  4. pMIEG3-Shp-2E76K plus pCD4;

  5. pMIEG3-Shp-2E76K plus pCD4-GATA2;

  6. pMIEG3-Shp-2E76K plus pCD4-ΔGATA2.

Cells were stained with anti-human CD4 conjugated to allophycocyanin (APC), sorted for EGFP+APC+ cells, and plated into progenitor assays. Colonies were scored for colony forming unit (CFU)-granulocyte-macrophage (GM), monocyte (M), granulocyte (G), and granulocyte-erythroid-monocyte-megakaryocyte (GEMM). As predicted, cells co-transduced with activating Shp2 mutant E76K and a secondary empty vector produced significantly more CFU-M than cells expressing WT Shp2. Upon co-transduction with GATA2, the number of CFU-M generated from E76K-expressing cells was significantly reduced. In contrast, cotransduction of ΔGATA2 lacking the C-terminal zinc finger failed to normalize the number of CFU-M produced by E76K-expressing cells. Mechanistically, we reasoned that if the c-Jun-PU.1 interaction contributes to the mutant Shp2-induced aberrant monocytic differentiation, we would observe increased expression directed by the macrophage colony-stimulating factor receptor (MCSFR) promoter, originally characterized to be activated by PU.1 and co-activator c-Jun. Consistently, MCSFR promoter-directed luciferase expression exhibited higher levels in NIH3T3 cells expressing Shp2E76K compared to those expressing WT Shp2. Furthermore, co-immunoprecipitation assays using nuclear extracts demonstrate increased c-Jun-PU.1 complexes in mutant Shp2-expressing hematopoietic progenitors, while chromatin immunoprecipitation assays demonstrate an increased association of the c-Jun-PU.1 complex at the MCSFR promoter. Moreover, CD34+ JMML progenitors express significantly higher levels of c-JUN than CD34+ cord blood progenitors from healthy newborns, substantiating the disease relevance of these mechanistic findings. These data suggest that one fundamental sequela of Ras hyperactivation in mutant Shp2-expressing cells is elevated c-Jun expression and an increased c-Jun-PU.1 interaction promoting monocytic differentiation and, thus, overproduction of myelomonocytic cells, the hallmark of JMML. These findings imply that pharmaceutical interventions that inhibit c-Jun expression or function could theoretically inhibit mutant Shp2-induced monocytic differentiation and, thus, serve as a novel approach for treatment of JMML.

Disclosures: No relevant conflicts of interest to declare.

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